The Mad (Mothers against decapentaplegic) gene family is thought to play a key role in the cytoplasmic transduction of signals from the transforming growth factor beta (TGF-beta) family of ligands. The bone morphogenetic proteins (BMPs) are TGF-beta type ligands implicated in developmental processes which include patterning and differentiation of the vertebrate limb. This research proposed here will address the role of Mad proteins in the developing chick limb by examining the effect ectopic expression (by retroviral mediated gene transfer) of the human Mad proteins Smad1, Smad5, and DPC4. Dominant negative and constitutively activated forms of these proteins will also be tested. The position of Mad proteins in the BMP signal transduction pathway will be tested by assaying the ability of wild-type and mutant Mads to suppress the effects of activated and dominant negative versions of the BMP receptor. Finally, the role of endogenous Mads in the development of the chick embryonic limb will be confirmed by the cloning and characterization of chick Mads. This characterization will include in situ hybridization analysis as well as overexpression in the embryonic limb to confirm the phenotypes previously obtained with human Mads. The TGF- beta family of ligands have been implicated in the control of cell growth, and loss of the Mad genes DPC4 and MADR2 has been associated with certain carcinomas. Therefore the study of Mad function in a biological context, as outlined in this proposal, will lead to a greater understanding of the control of cell growth and development, critical processes in the genesis of tumors.